Insulin is a polypeptide hormone produced in the beta cells of the islets of Langerhans. The active insulin molecule is a two-chain molecule consisting of a B- and an A-Chain connected by two disulphide bridges. The insulin is synthesized as a precursor molecule proinsulin with the structure B-C-A wherein the C-peptide chain connects the C-terminal amino acid residue in the B-chain with the N-terminal amino acid residue in the A-chain. Mature two-chain insulin is formed by cleavage of the C-peptide at the pair of basic amino acid residues situated at the junctions with the A- and B-chain. The A- and B-chain are held together by two disulphide bridges between the A7 and B7 and the A20 and B19 Cys residues, respectively. In addition, the biologically active insulin molecule has an internal disulphide bridge between the Cys residues in the position A6 and A11.
After the development of recombinant DNA technology numerous methods have been described to produce insulin and precursors thereof in genetically modified host cells. Thus methods for making insulin from E. coli are disclosed in e.g Frank, B. H., Pettee, J. M., Zimmerman, R. E. & Burck, P. J. In: Peptides. Synthesis-Structure-Function. Proceedings of the Seventh American Peptide Symposium (D. H. Rich and E. Gross, eds). Pierce Chemical Company, p. 729 (1981). As E. coli does not have the cellular machinery for folding the expressed polypeptide and establish the disulphide bridges connecting the A- and B chain in the mature insulin this strategy includes a number of in vitro processing steps such as in vitro establishment of the disulphide bridges during refolding and subsequent cleavage of the C-peptide.
In contrast to E. coli eukaryotes contain the necessary machinery for folding and establishing disulphide bridges and thus would seem to be good candidates for production of mature insulin in genetically modified organisms. U.S. Pat. No. 4,914,026 discloses a process for making mature insulin in yeast by insertion of the human proinsulin gene linked to the yeast α-factor leader sequence in a yeast host cell and growing the transformed yeast cell in a nutrient medium under conditions whereby proinsulin is expressed and secreted in mature form.
Thim et al, Proc Natl. Acad. Sci. USA, volume 83, 6766-6770 discloses expression of human proinsulin and a number of insulin precursors with a modified C-peptide such as RREAENLQKR (SEEQ ID NO:1), RREAPLQKR (SEQ ID NO:2), RREALQKR (SEQ IDNO:3), KREALQKR (SEQ ID NO:4) and RRLQKR (SEQ ID NO:5). SEQ ID NO:5 is also disclosed by Thim et al, in FEBS Letters, volume 212, number 2, 307-312.
Furthermore, WO 97/03089 disclosed expression of insulin precursors with the formula BZA wherein B and A are the A and B peptide chains of human insulin being linked by at least one disulphide bond and Z is a polypeptide comprising at least one proteolytic cleavage site, e.g. KREQKLISEEALVDKR (SEQ ID NO:6).
However, the disclosed insulin precursors only give rise to minute amounts of secreted mature insulin in the culture medium.
European patent application, 0163529A, PCT patent applications Nos. WO 95/02059 and WO 90/10075 disclose processes for making insulin and insulin analogues based on expression of a precursor of the insulin or insulin analogue in yeast that following initial recovery from the fermentation broth are enzymatically converted to the mature insulin or insulin analogue. The precursor molecules comprise certain modified C-peptides and may furthermore comprise an N-terminal extension of the insulin B-chain. The modified C-peptide and the possible N-terminal extension of the B-peptide are designed not to be cleaved in the yeast cell and thus the precursors are secreted as single chain peptides wherein the A- and the B-chain are still connected by the modified C-peptide but with correctly positioned disulphide bridges. The mature insulin or insulin analogue product is then obtained by a number of subsequent in vitro enzymatic steps to cleave the C-peptide and possibly the N-terminal extension. These enzymatic steps are time consuming, often costly and introduce additional impurities that subsequently have to be removed in further downstream process steps like expensive chromatography steps and the like.
A process for making mature insulin in genetically engineered animal cells that are not naturally capable of forming secretory granules is disclosed in U.S. Pat. No. 6,348,327.
The purpose of the present invention is to develop a fungi strain capable of secreting fully processed mature human insulin analogues so that expensive and time consuming downstream purification process steps are avoided.